Growth factors are polypeptides which stimulate a wide variety of biological responses (e.g. DNA synthesis, cell division, expression of specific genes, etc.) in a defined population of target cells. A variety of growth factors have been identified, including the transforming growth factor beta family (TGF-βs), epidermal growth factor and transforming growth factor alpha (the TGF-αs), the platelet-derived growth factors (PDGFs), the fibroblast growth factor family (FGFs) and the insulin-like growth factor family (IGFs), which includes IGF-I and IGF-II. Many growth factors have been implicated in the pathogenesis of cancer.
IGF-I and IGF-II (the “IGFs”) are related in amino acid sequence and structure, with each polypeptide having a molecular weight of approximately 7.5 kilodaltons (kDa). IGF-I mediates the major effects of growth hormone, and is thus the primary mediator of growth after birth. IGF-I has also been implicated in the actions of various other growth factors, since the treatment of cells with such growth factors leads to increased production of IGF-I. In contrast, IGF-II is believed to have a major role in fetal growth. Both IGF-I and IGF-II have insulin-like activities (hence their names), and are mitogenic (stimulate cell division).
IGF-I has been found to stimulate the growth of cells from a number of different types of cancer (Butler et al., 1998 Cancer Res. 58(14):3021-3027; Favoni R E, et al., 1998, Br. J. Cancer 77(12): 2138-2147). Additionally, IGF-I has additionally been found to exert anti-apoptotic effects on a number of different cell types, including tumor cells (Giuliano M, et al., 1998 Invest Ophthalmol. Vis. Sci. 39(8): 1300-1311; Zawada W M, et al., 1998, Brain Res. 786(1-2): 96-103; Kelley K W, et al., 1998, Ann. N. Y. Acad. Sci. 840: 518-524; Toms S A, et al., 1998, J. Neurosurg. 88(5): 884-889; Xu F, et al., 1997, Br. J. Haematol. 97(2): 429-440). Prospective studies have implicated IGF-I as a risk factor for cancers of the prostate, breast, and colon, while IGFBP-3, the major circulatory binding protein for IGFs, appears to have a protective effect. A variety of other observations further support the idea that the relative balance of IGFBP-3 to other IGF-binding proteins (notably IGFBP-2) is somehow instrumental in the control of tumor cell growth, both in vitro and in vivo. Recent evidence also suggests that IGFBP-3 may play a central role in the growth and apoptosis of tumor cells in an IGF-independent manner.
Approximately half of the 1.3 million patients diagnosed with cancer each year in the U.S. have (or will be at risk for) systemic disease. Chemotherapy is the most common therapeutic approach for these patients. Most chemotherapeutic agents are effective primarily against dividing cells, and myelosuppression is often the dose-limiting toxicity. Chemical agents fall into several categories and have different mechanisms of action but, at effective doses, most have side-effects which seriously impact the patient's quality of life. doxorubicin (ADRIAMYCIN®), irinotecan (CPT-11), paclitaxel (TAXOL®), cisplatin, tamoxifen, methotrexate and 5-fluorouracil are popular agents used to treat a variety of cancers, sometimes in combination. In addition to myelosuppression, gastrointestinal effects, mucositis, alopecia, and (in the case of doxorubicin) cardiac toxicities are also observed with these agents.
Clearly, it would be of interest to find ways to make tumor cells selectively sensitive to these chemical agents. One approach might be to target the very properties that make cancer cells unique. Cancer cells generally evolve strategies for circumventing the normal cell cycle checkpoint controls that target cells for self-destruction after sustaining the kind of DNA damage typically inflicted by chemotherapeutic agents. If such functions could be even partially restored in tumor cells by pretreatment with a “sensitizing” agent, one would predict that such treatment would exert a selective effect on such cells.
Almost all IGF circulates in a non-covalently associated complex of IGF-I, insulin-like growth factor binding protein 3 (IGFBP-3) and a larger protein subunit termed the acid labile subunit (ALS), such that very little free IGF-I is detectable. The ternary complex is composed of equimolar amounts of each of the three components. ALS has no direct IGF-binding activity and appears to bind only to the IGF/IGFBP-3 complex (Baxter et al., J. Biol. Chem. 264(20):11843-11848, 1989), although some reports suggest that IGFBP-3 can bind to rat ALS in the absence of IGF (Lee et al., Endocrinology 136:4982-4989, 1995). The ternary complex of IGF/IGFBP-3/ALS has a molecular weight of approximately 150 kDa and has a substantially increased half-life in circulation when compared to binary IGF/IGFBP-3 complex or IGF alone (Adams et al., Prog. Growth Factor Res. 6(2-4):347-356; presented October 1995, published 1996). This ternary complex is thought to act “as a reservoir and a buffer for IGF-I and IGF-II preventing rapid changes in the concentration of free IGF” (Blum et al. (1991), “Plasma IGFBP-3 Levels as Clinical Indicators” in MODERN CONCEPTS OF INSULIN-LIKE GROWTH FACTORS, pp. 381-393, E. M. Spencer, ed., Elsevier , New York). While there is essentially no excess (unbound) IGFBP-3 in circulation, a substantial excess of free ALS does exist (Baxter, J. Clin. Endocrinol. Metab. 67:265-272, 1988).
How IGFBP-3 mediates its cellular effects is not well understood, although there is indirect evidence to suggest that it mediates some of the effects of p53, a well-characterized tumor suppressor (Ferry et al., (1999) Horm Metab Res 31(2-3):192-202). IGFBP-3 is mobilized to the nucleus of rapidly growing cells (Schedlich, et al., (1998) J. Biol. Chem. 273(29):18347-52; Jaques, et al., (1997) Endocrinology 138(4):1767-70). A useful step toward defining the functional interactions of IGFBP-3 would be to identify protein domains involved in the ability of IGFBP-3 to specifically bind a surprisingly large array of intracellular and extracellular targets. Known targets include: IGF-I, IGF-II, insulin (under some conditions), acid-labile subunit (ALS), plasminogen, fibrinogen, transferrin, lactoferrin, collagen Type Ia, prekallikrein, RXR-alpha, viral oncoproteins, heparin, specific proteases, cellular receptors, a number of intracellular targets identified in two-hybrid screens, and components of the nuclear localization transport machinery (Mohseni-Zadeh and Binoux (1997) Endocrinology 138(12):5645-8; Collett-Solberg, et al. (1998) J. Clin. Endocrinol Metab. 83(8):2843-8; Rajah, et al. (1995) Prog. Growth Factor Res. 6(2-4):273-84; Fowlkes and Serra (1996) J. Biol. Chem. 271:14676-14679; Campbell, et al. (1999) J. Biol Chem. 274(42):30215-21; Durham, et al. (1999) Horm Metab Res 31(2-3):216-25; Campbell, et al. (1998) Am J Physiol. 275(2Pt 1):E321-31). A better understanding of these binding interactions might allow the generation of IGFBP-3 variants lacking one or more of these functions. The activity of these and other variants in in vivo models may suggest novel therapeutic strategies based either on the variant proteins themselves, mimetics, or small organic molecules selected from combinatorial chemistry libraries created with the information gained from a study of these variants.
A recently described mutant in which residues 228-232 of IGFBP-3 have been substituted with the corresponding residues from IGFBP-1 (a closely related protein) shows impaired binding to ALS, RXR-alpha, and plasminogen (Campbell, et al. (1998) Am. J. Physiol. 275(2 Pt 1):E321-31; Firth, et al. (1998) J. Biol. Chem. 273:2631-2638). Specific proteolysis of IGFBP-3 under certain physiological conditions such as pregnancy and critical illness can lead to altered binding and release of its IGF ligand. The binary complex of IGFBP-3 with IGF-I or IGF-II (both growth factors bind IGFBP-3, with similar affinities) can extravasate across endothelial junctions to the intercellular milieu where IGFBP-3 can interact specifically with glycosaminoglycans, specific proteases, and cell-surface proteins.
It should be noted that, while IGFBP-3 is the most abundant of the IGF binding proteins (“IGFBPs”), at least five other distinct IGFBPs have been identified in various tissues and body fluids. Although these proteins bind IGFs, they originate from separate genes and have distinct amino acid sequences. Unlike IGFBP-3, other circulating IGFBPs are not saturated with IGFs. IGFBP-3 and IGFBP-5 are the only known IGFBPs which can form the 150 kDa ternary complex with IGF and ALS. The IGF and ALS binding domains of IGFBP-3 are thought to be in the N-terminal portion of the protein, as N-terminal fragments of the protein isolated from serum retain these binding activities. However, some of the other IGFBPs have also been suggested for use in combination with IGF-I as therapeutics.
In addition to its role as the major carrier protein for IGF in serum, IGFBP-3 has been recently shown to have a number of different activities. IGFBP-3 can bind to an as-yet unidentified molecule on the cell surface, where it can inhibit the activity of exogenously-added IGF-I (Karas et al., 1997, J. Biol. Chem. 272(26):16514-16520). Although the binding of IGFBP-3 to cell surfaces can be inhibited by heparin, the unidentified cell surface binding molecule is unlikely to be a heparin-like cell surface glycosaminoglycan, because enzymatic removal of heparin glycosaminoglycans has no effect on IGFBP-3 cell surface binding (Yang et al., 1996, Endocrinology 137(10):4363-4371). It is not clear if the cell surface binding molecule is the same or different than the IGFBP-3 receptor that was identified by Leal et al. (1997, J. Biol. Chem. 272(33):20572-20576), which is identical to the type V transforming growth factor-beta (TGF-β) receptor.
IGFBP-3, when used alone in in vitro assays, has also been reported to promote apoptosis. Interestingly, IGFBP-3 has been shown to promote apoptosis in cells with and without functional type 1 IGF receptors (Nickerson et al., 1997, Biochem. Biophys. Res. Comm. 237(3):690-693; Rajah et al., 1997, J. Biol. Chem. 272(18):12181-12188). However, there are conflicting reports as to whether apoptosis is induced by full length IGFBP-3 or a proteolytic fragment of IGFBP-3 (Rajah et al., ibid; Zadeh et al., 1997, Endocrinology 138(7):3069-3072). More recently, a wealth of unpublished data gathered in a number of laboratories fails to support some of the claims made in the above publications. In in vivo models tested to date, infused IGFBP-3 protein alone has showed mixed results in limiting tumor growth.
U.S. Pat. No. 5,681,818 claims the administration of IGFBP-3 for controlling the growth of somatomedin dependent tumors in the treatment of cancer. U.S. Pat. No. 5,840,673 also describes the indirect intracellular modulation of IGFBP-3 levels as a method for controlling tumor growth. U.S. Pat. No. 6,015,786 discloses the use of IGFBP-3 complexed with mutant IGF for the treatment of IGF-dependent tumors. However, each of these patents discloses a direct in vivo effect of administered IGFBP-3 protein on tumor growth. None of these patents envisages a situation where IGFBP-3 has no effect on tumors on its own, yet sensitizes tumors to the action of other agents. Numerous publications (Williams, et al., Cancer Res 60(1):22-7, 2000; Perks, et al., J Cell Biochem 75(4):652-64, 1999; Maile et al., Endocrinology 140(9):4040-5, 1999; Gill, et al., J Biol Chem 272(41):25602-7, 1997) further demonstrate combined effects of IGF binding proteins, radiation and ceramide on cultured cells. However, it is difficult or impossible to extrapolate from tissue culture results to effectiveness in vivo. In one report (Portera et al, Growth Hormone & IGF Research 2000, Supplement A, S49-S50, 2000) IGFBP-3 combined with CPT-11 showed additive effects in a colon cancer model both in vivo and in vitro, but IGFBP-3 alone also showed effects on tumor growth in this model. At the present time, a widely held belief among skilled practitioners in the field is that IGFBP-3 alone may sometimes control tumor growth directly. No one has shown that systemically administered IGFBP-3 can sensitize tumor cells in animals to the action of co-administered agents, without inhibiting tumor growth when used on its own at similar doses.
Such a distinction is of considerable practical importance. Among other things, it means that many types of tumors that may appear to be recalcitrant to IGFBP-3 treatment when used singly, or to some other agent used on its own, may in fact be quite susceptible to the combination. Unless this fact is appreciated, even the testing of certain combinations of substances which, on their own, are known to have no effects on tumor growth, may never be undertaken.
IGF-I and IGFBP-3 may be purified from natural sources or produced by recombinant means. For instance, purification of IGF-I from human serum is well known in the art (Rinderknecht et al. (1976) Proc. Natl. Acad. Sci. USA 73:2365-2369). Production of IGF-I by recombinant processes is shown in EP 0 128 733, published in December of 1984. IGFBP-3 may be purified from natural sources using a process such as that shown in Baxter et al. (1986, Biochem. Biophys. Res. Comm. 139:1256-1261). Alternatively, IGFBP-3 may be synthesized by recombinantly as discussed in Sommer et al., pp. 715-728, MODERN CONCEPTS OF INSULIN-LIKE GROWTH FACTORS (E. M. Spencer, ed., Elsevier, New York, 1991). Recombinant IGFBP-3 binds IGF-I in a 1:1 molar ratio.
Topical administration of IGF-I/IGFBP-3 complex to rat and pig wounds is significantly more effective than administration of IGF-I alone (Id.). Subcutaneous administration of IGF-I/IGFBP-3 complex to hypophysectomized, ovariectomized, and normal rats, as well as intravenous administration to cynomolgus monkeys, “substantially prevents the hypoglycemic effects” of IGF-I administered alone (Id.).
The use of IGF/IGFBP-3 complex has been suggested for the treatment of a wide variety of disorders (see, for example, U.S. Pat. Nos. 5,187,151, 5,527,776, 5,407,913, 5,643,867, 5,681,818 and 5,723,441, as well as International Patent Applications Nos. WO 95/03817, WO 95/13823, and WO 96/02565. IGF-I/IGFBP-3 complex is also under development by Insmed Pharmaceuticals, Inc., as a treatment for several indications, including diabetes and recovery from hip fracture surgery.
For practitioners skilled in the art, the complex of IGF-I and IGFBP-3 is generally considered to be a different compound, and to have different biological effects, than IGFBP-3 alone.
While there are a large number of cytotoxic drugs available for the treatment of cancer, these drugs are generally associated with a variety of serious side effects, including alopecia, leukopenia, mucositis. Accordingly, there is a need in the art for cancer therapies that do not induce the serious side effects associated with conventional cytotoxic chemotherapy. One method for achieving this goal is to make target cells (such as tumor cells) selectively sensitive to cytotoxic drugs, thereby permitting the effective use of such drugs at lower doses not associated with serious side effects.
A number of reports claim IGFBP-3 alone can cause apoptosis in tumor cells in culture, and others have described additive effects of combining IGFBP-3 treatment with various chemical agents in tissue culture (cited above). However, it is unclear how these effects relate, if at all, to in vivo models.
The key assumption in all of the above examples is that the efficacy of combination treatments involving administration of IGFBP-3 and other agents is investigated only after IGFBP-3 has shown efficacy on its own. Given this state of thinking, which is established in the field at this time, it is therefore unlikely that effective combination regimens will be identified unless IGFBP-3 alone is shown to have efficacy in the first place. Synergistic effects with co-administered agents showing marginal efficacy themselves would be even harder to identify.
Herceptin, a humanized antibody used in the treatment of breast cancer, has exemplified the use of large proteinaceous molecules to extend the therapeutic efficacy of chemical agents (Pegram, et al. (1998) J. Clin. Oncol. 16(8):2659-71). However, this molecule was approved for clinical use based on its own efficacy on tumors and survival, when used alone. Additive effects have been observed when this molecule is administered in combination with chemical agents.